Control of bioreactor processes

ABSTRACT

Processes, as well as associated systems and computer program (software) products, are disclosed for the biological conversion of CO into desired end products such as ethanol. The control methodologies used for these processes can advantageously result in a reduced time required for a batch operation or other initial operating period, prior to achieving a continuous operation, which may be demarcated either by the addition of fresh culture medium at a defined flow rate or by another process initiation target. The control methodologies may alternatively, or in combination, improve a process performance parameter, such as productivity of the desired end product or bacterial growth rate, during this batch operation or other initial operating period.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a Continuation of Co-pending U.S. application Ser.No. 14/329,881 filed on Jul. 11, 2014 the contents of which areincorporated by reference.

FIELD OF THE INVENTION

Aspects of the invention relate to the initiation of processes for themicrobial fermentation of CO-containing substrates to ethanol, forexample to achieve continuous and steady-state operation. Specificaspects relate to the manner in which operating parameters arecontrolled, leading to advantageous results.

DESCRIPTION OF RELATED ART

Environmental concerns over fossil fuel greenhouse gas (GHG) emissionshave led to an increasing emphasis on renewable energy sources. As aresult, ethanol is rapidly becoming a major hydrogen-rich liquidtransport fuel around the world. Continued growth in the global marketfor the fuel ethanol industry is expected for the foreseeable future,based on increased emphasis in ethanol production in Europe, Japan, andthe United States, as well as several developing nations. For example,in the United States, ethanol is used to produce E10, a 10% mixture ofethanol in gasoline. In E10 blends, the ethanol component acts as anoxygenating agent, improving the efficiency of combustion and reducingthe production of air pollutants. In Brazil, ethanol satisfiesapproximately 30% of the transport fuel demand, as both an oxygenatingagent blended in gasoline, and as a pure fuel in its own right. Inaddition, the European Union (EU) has mandated targets, for each of itsmember nations, for the consumption of sustainable transport fuels suchas biomass-derived ethanol.

The vast majority of fuel ethanol is produced via traditionalyeast-based fermentation processes that use crop derived carbohydrates,such as sucrose extracted from sugarcane or starch extracted from graincrops, as the main carbon source. However, the cost of thesecarbohydrate feed stocks is influenced by their value in the marketplacefor competing uses, namely as food sources for both humans and animals.In addition, the cultivation of starch or sucrose-producing crops forethanol production is not economically sustainable in all geographies,as this is a function of both local land values and climate. For thesereasons, it is of particular interest to develop technologies to convertlower cost and/or more abundant carbon resources into fuel ethanol. Inthis regard, carbon monoxide (CO) is a major, energy-rich by-product ofthe incomplete combustion of organic materials such as coal, oil, andoil-derived products. CO-rich waste gases result from a variety ofindustrial processes. For example, the steel industry in Australia isreported to produce and release into the atmosphere over 500,000 metrictons of CO annually.

More recently, micro-organism (bacterial) based process alternatives forproducing ethanol from CO on an industrial scale have become a subjectof commercial interest and investment. The ability of micro-organismcultures to grow, with CO being the sole carbon source, was firstdiscovered in 1903. This characteristic was later determined to residein an organism's use of the acetyl coenzyme A (acetyl CoA) biochemicalpathway of autotrophic growth (also known as the Woods-Ljungdahl pathwayand the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS)pathway). A large number of anaerobic organisms includingcarboxydotrophic, photosynthetic, methanogenic, and acetogenic organismshave since been shown to metabolize CO. Anaerobic bacteria, such asthose from the genus Clostridium, are known to produce ethanol from CO,CO₂ and H₂ via the acetyl CoA biochemical pathway. For example, variousstrains of Clostridium ljungdahlii that produce ethanol from gases aredescribed in WO 00/68407; EP 1117309 A1; U.S. Pat. No. 5,173,429; U.S.Pat. No. 5,593,886; U.S. Pat. No. 6,368,819; WO 98/00558; and WO02/08438. The bacterium Clostridium autoethanogenum sp is also known toproduce ethanol from gases (Abrini et al., ARCHIVES OF MICROBIOLOGY 161:345-351 (1994)).

Because each enzyme of an organism promotes its designated biologicalconversion with essentially perfect selectivity, microbial synthesisroutes can achieve higher yields with lower energy costs compared toconventional catalytic routes. For example, the energy requirements forseparating byproducts, which result from non-selective side reactions,from the desired products may be reduced. In addition, concerns over thepoisoning of catalysts, due to impurities in the reaction medium, arediminished.

Despite these apparent advantages, however, the art must address certainchallenges associated with microbial synthesis of ethanol from CO,especially in terms of ensuring that the production rate is competitivewith other technologies. When using CO as their carbon source, theanaerobic bacteria described above produce ethanol by fermentation, butthey also produce at least one metabolite, for example CO₂, H₂, methane,n-butanol, and/or acetic acid. The formation of any of these metaboliteshas the potential to significantly impact productivity and overalleconomic viability of a given process, as available carbon is lost tothe metabolite(s) and the production efficiency of the desired endproduct is compromised. In addition, unless a metabolite (e.g., aceticacid) itself has value at the time and place of the microbialfermentation process, it may pose a waste disposal problem. Variousproposals for addressing the formation of products other than thedesired end product in the anaerobic fermentation of CO-containing gasesto make ethanol are discussed in WO2007/117157, WO2008/115080 andWO2009/022925.

Ethanol production rate, which is a key determinant as to whether agiven fermentation process is economically attractive, is highlydependent on managing the appropriate conditions for bacterial growth.For example, it is known from WO2010/093262 that the CO-containingsubstrate must be provided to a microbial culture at a rate that resultsin optimal microbial growth and/or desired metabolite production. Ifinsufficient substrate is provided, microbial growth slows and thefermentation product yields shift toward acetic acid at the expense ofethanol. If excessive substrate is provided, poor microbial growthand/or cell death can result. Further information regarding therelationships among operating parameters in these processes is found inWO2011/002318.

The control of operating parameters is particularly important during theinitial period of operation, in which the processing objectives arefocused on not only growing the cell culture to a sufficient level andestablishing other conditions for continuous operation, but alsobalancing the product and byproduct productivities. Reducing the timeneeded for conducting a batch culture operation, prior to continuousbioreactor operation, has major implications for improving processeconomics. This is particularly true in view of the fact that microbescapable of growing on CO-containing gases generally do so at a slowerrate than microbes used in competing technologies with sugars as a foodsource. From the commercial perspective of operating a fermentationprocess, the time required for a microbial population to becomeestablished, i.e., to reach a sufficiently high cell density for thesynthesis of economically favorable levels of product, represents a keyoperating cost affecting the overall profitability. The ability toenhance culture growth rates and/or productivities during an initialoperating period, for example under batch conditions, and thereby reducethe time required to reach desired cell densities and/or product levels,is an important determinant for overall success in the commercializationof biological processes for producing ethanol from CO-containing wastegas.

SUMMARY OF THE INVENTION

Aspects of the invention relate to methods for controlling theinitiation of biological CO conversion processes, based on availabledata. Normally, at the beginning of such processes, the bioreactor ischarged (inoculated) with a culture medium containing carboxydotrophicbacteria (i.e., having the ability to derive energy from CO). Accordingto representative processes, ethanol is the desired end product, whereasacetate is generated as an undesired metabolite, in the form of aceticacid. As discussed above, CO must be supplied judiciously to thebioreactor to meet competing objectives. In particular, an undersupplyof CO can result in excessive acetate formation at the expense ofethanol, whereas an oversupply of CO can negatively impact bacterialgrowth. In view of these considerations, a specified profile for theflow rate over time of CO or CO-containing gas may be used, based on theexpected bacterial growth during batch operation, in conjunction withinformation derived from other processes.

The overriding operating objective during an initial operating period(e.g., a batch operation period) is to increase the concentration ofbacteria (biomass), in the culture medium. Therefore, the gas flowprofile during the batch operation period is normally conservative andseeks to avoid the oversupply of CO. This can result in the formation ofacetic acid in a significant amount, in some cases exceeding that of thedesired ethanol end product. Because any acetic acid that is generatedthroughout the bacterial conversion processes lowers the pH value of theculture medium, a basic neutralizing agent such as aqueous ammoniumhydroxide may be introduced. The neutralizing agent may be dosed to thebioreactor to maintain a pH value (e.g., a pH of 5.0) of the culturemedium suitable for bacterial growth.

Embodiments of the invention are directed to biological fermentationprocesses for converting CO into a desired end product such as ethanol,comprising feeding both a CO-containing substrate and a basicneutralizing agent (e.g., aqueous ammonium hydroxide) to a bioreactorcomprising a culture medium containing carboxydotrophic bacteria. Theprocesses generate both the desired end product as well as an acidicmetabolite (e.g., acetic acid) that is converted by the neutralizingagent (e.g., to a salt such as ammonium acetate), in order to avoidunacceptable pH levels in the culture medium. According to onerepresentative embodiment, the flow rate of the basic neutralizing agentmay be controlled based on a measured property, such as a measuredconcentration or measured productivity of the carboxydotrophic bacteriaor acidic metabolite, in the culture medium. Alternatively, if suchmeasured property is unavailable, for example, if suitable on-linesampling and analytical equipment are lacking, the flow rate of thebasic neutralizing agent may be controlled based on a measured flow rateof the CO-containing substrate or otherwise based on a set point forthis substrate.

Other embodiments of the invention are directed to systems comprising abioreactor and a controller configured to control the flow rate of thebasic neutralizing agent to the bioreactor, based on either a measuredproperty of the culture medium as described above or, alternatively,based on a measured flow rate of the CO-containing substrate orotherwise based on a set point for this substrate. In the case ofcontrol based on a measured property of the culture medium, the systemmay further comprise the necessary sampling apparatus, configured toisolate a sample of the culture medium from the bioreactor for analysis,in addition to an analyzer configured to analyze the isolated sample. Ineither of the above control method alternatives, representative systemsmay optionally comprise a second controller configured to control aCO-containing substrate flow rate based on a measured pH value, asampling apparatus configured to isolate, from the bioreactor, a sampleof the culture medium, and/or an analyzer configured to analyze thesample and then input, to the controller, the measured pH value.

Further embodiments of the invention are directed to computer programproducts comprising non-transitory computer readable media havingcomputer programs embodied thereon. These computer programs includeinstructions for causing a processor to perform steps needed to carryout the control processes described herein. These processes includereceiving information that is input to a controller configured tocontrol a basic neutralizing flow rate to a bioreactor. The informationthat may be received and input, in this manner, includes informationreceived from an analyzer configured to analyze a culture medium samplefrom the bioreactor for a measured property as described above.Alternatively, the information may be the measured flow rate of theCO-containing substrate, received from a flow rate sensor or measurementdevice that is configured to measure this flow. The received informationmay also include a CO-containing substrate flow rate set point.Regardless of the type of information that is received and input to acontroller, representative processes may further comprise receiving ameasured pH value, for example, from a pH meter or other analyzerconfigured to measure the pH of the culture medium directly or otherwisea sample of the culture medium from the bioreactor. The measured pHvalue may be input to a second controller configured to control theCO-containing substrate flow rate, whereby the measured pH value is thebasis for control.

These and other embodiments and aspects relating to the presentinvention are apparent from the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the exemplary embodiments of thepresent invention and the advantages thereof may be acquired byreferring to the following description in consideration of theaccompanying figures.

FIG. 1 is a flow chart of a representative methodology for controllingoperating parameters of a biological process for converting aCO-containing substrate to ethanol.

FIG. 2 is a graph of measured concentrations of ethanol,carboxydotrophic bacteria, and acetic acid, in a culture medium overtime, for a biological process for converting a CO-containing substrateto ethanol, using a conventional control methodology.

FIG. 3 is a graph of measured concentrations of ethanol,carboxydotrophic bacteria, and acetic acid in a culture medium overtime, for a biological process for converting a CO-containing substrateto ethanol, using a control methodology as described herein.

FIG. 4 is a comparative graph of the CO-containing substrate flow rateover time, for biological processes for converting a CO-containingsubstrate to ethanol, using a conventional control methodology and acontrol methodology as described herein.

FIG. 5 is a comparative graph of the carboxydotrophic bacteriaconcentration in a culture medium over time, for biological processesfor converting a CO-containing substrate to ethanol, using aconventional control methodology and a control methodology as describedherein.

FIG. 6 is a graph of measured concentrations of ethanol,carboxydotrophic bacteria, and acetic acid, in a culture medium overtime, as well as the measured flow rate of fresh culture medium, for abiological process for converting a CO-containing substrate to ethanol,using a representative control methodology as described herein.

FIG. 7 is a graph of measured concentrations of ethanol,carboxydotrophic bacteria, and acetic acid in a culture medium overtime, as well as the measured flow rates of NH₄OH neutralizing agentsolution and CO-containing substrate, for a biological process forconverting a CO-containing substrate to ethanol, using an alternativecontrol methodology as described herein.

DETAILED DESCRIPTION

The present invention relates to processes for producing a desired endproduct, such as ethanol, by feeding CO in a CO-containing substrate toa bioreactor comprising a culture medium containing carboxydotrophicbacteria. In addition to the desired end product, representativeprocesses additionally generate undesired or less desired metabolites.An example of an acidic metabolite that may be generated in addition toa desired product, such as ethanol, is acetate (e.g., in the form ofacetic acid). Representative carboxydotrophic bacteria or microbes(i.e., microorganisms that obtain energy and carbon from CO), are thosefrom the genus Moorella, Clostridia, Ruminococcus, Acetobacterium,Eubacterium, Butyribacterium, Oxobacter, Methanosarcina, Methanosarcina,and Desulfotomaculum. Particular examples of bacteria that areClostridia include C. ljundahlii, C. autoethanogenum, C. ragsdalei, andC. beijerenckei.

Representative CO-containing substrates include broadly anyCO-containing gas, or possibly liquid, in which carbon monoxide can bemade available to one or more strains of bacteria for growth and/orfermentation. Such CO-containing substrates preferably do not includecontaminants to the extent that such contaminants might have an adverseeffect on the growth of the carboxydotrophic bacteria (e.g., one or morecontaminant(s) are not present in concentrations or amounts such thatthe growth rate is reduced by more than 10% under a given set ofconditions, compared to the growth rate under the same conditions, butwithout the contaminant(s)). Representative gaseous CO-containingsubstrates typically contain a significant proportion of CO, preferablyat least about 5% to about 100% CO by volume. Such substrates are oftenproduced as waste products of industrial processes such as steelmanufacturing processes or non-ferrous product manufacturing process.Other processes in which gaseous CO-containing substrates are generatedinclude the gasification of organic matter such as methane, ethane,propane, coal, natural gas, crude oil, low value residues from oilrefinery (including petroleum coke or petcoke), solid municipal waste orbiomass. Biomass includes by-products obtained during the extraction andprocessing of foodstuffs, such as sugar from sugarcane, or starch frommaize or grains, or non-food biomass waste generated by the forestryindustry. Any of these carbonaceous materials can be gasified, i.e.partially combusted with oxygen, to produce synthesis gas (syngascomprising significant amounts of H₂ and CO). Advantageously, gasstreams from these processes may be used as described herein for thebeneficial production of useful end products such as ethanol. In otherembodiments, the substrate comprising CO can be derived from the steamreforming of hydrocarbons. These processes are described in more detailin U.S. Application publication Nos. U.S.2013/0045517A1;U.S.2013/0210096A1; U.S.2013/0203143A1 and U.S.2013/0316411A1 and U.S.Pat. No. U.S. Pat. No. 8,383,376 the contents of all of which areincorporated by reference in their entirety.

While it is not necessary for the CO-containing substrate to contain anyhydrogen, the presence of H₂ is normally not detrimental to theformation of the desired end product. In particular embodiments, theCO-containing substrate may comprise low concentrations of H₂, forexample, less than 10% by volume, less than 5% by volume, or less than1% by volume. The CO-containing substrate may also contain some CO₂, forexample, from about 1% to about 80% by volume, from about 1% to about50% by volume, or from 1% to about 30% by volume. Any CO-containingsubstrate, such as a gaseous CO-containing substrate, may be treated toremove any undesired impurities, such as dust particles or any othersolid, liquid, or gaseous contaminants that may be detrimental to thecarboxydotrophic bacteria or the biological conversion process ingeneral, prior to its use in the biological conversion process. Forexample, the gaseous CO-containing substrate may be filtered or scrubbedusing known methods.

In the context of an acidic metabolite that is acetic acid, the terms“acetic acid” or “acetate” refer to the total acetate present in theculture medium, either in its anionic (dissociated) form (i.e., asacetate ion or CH₃COO⁻) or in the form of free, molecular acetic acid(CH₃COOH), with the ratio these forms being dependent upon the pH of thesystem. The term “bioreactor” includes any suitable vessel forcontaining a culture of carboxydotrophic bacteria that may be used tocarry out the biological processes described herein, which may also bereferred to as fermentation processes, to the extent that they aregenerally conducted anaerobically. A suitable bioreactor may be aContinuous Stirred Tank Reactor (CSTR), an Immobilized Cell Reactor(ICR), a Trickle Bed Reactor (TBR), a Moving Bed Biofilm Reactor (MBBR),a Bubble Column, a Gas Lift Fermenter, a Membrane Reactor such as HollowFiber Membrane Bioreactor (HFMBR), a Static Mixer, or may include othervessels or devices (e.g., towers or piping arrangements) suitable forcontacting the CO-containing substrate with the bacterial culture medium(e.g., with dissolution and mass transport kinetics favorable forcarrying out the biological conversion).

Other suitable process streams, operating parameters, and equipment foruse in the biological processes described herein are described in U.S.patent application Publication No. U.S.2011/0212433, which is herebyincorporated by reference in its entirety.

The present invention is more particularly associated with the discoveryof biological processes for converting CO to valuable end products suchas ethanol, in which (i) the time required for a batch operation periodor other initial operating period, prior to achieving a continuousoperation, which may be demarcated either by the addition of freshculture medium at a defined flow rate or by another process initiationtarget, is unexpectedly reduced and/or (ii) productivity of the desiredend product or another process performance parameter (e.g., bacterialgrowth rate) is unexpectedly improved during this batch operation periodor other initial operating period. The conversion from batch operationto continuous operation may be demarcated by the commencement of addingfresh culture medium to the bioreactor used in the process.Alternatively, if the rate of fresh culture medium addition is increasedgradually rather than commenced at a discreet time point, the conversionfrom batch to continuous operation may be demarcated by achieving atarget rate of fresh culture medium addition to, and/or achieving atarget rate of bacteria-containing culture medium withdrawal from, thebioreactor. The target rates of fresh culture medium addition and/orbacteria-containing culture medium withdrawal may be the ratesassociated with a steady-state operation, i.e., an operation under whichconditions are held substantially constant over an extended period(e.g., at least about 3 days, or at least about 10 days) of productionof a desired end product. Otherwise, these target rates may be at leastabout 60%, at least about 75%, or at least about 90% of the ratesassociated with steady-state operation.

Aside from a target rate of fresh culture medium, other processinitiation targets that may be used to demarcate an initial operatingperiod from a steady-state or “on-stream” operating period can include aculture medium concentration of desired product (e.g., ethanol),carboxydotrophic bacteria, or acidic metabolite. Process initiationtargets may also include a productivity of desired product,carboxydotrophic bacteria, or acidic metabolite. Process initiationtargets may be predetermined, i.e., established from the outset of theprocess and possibly used as inputs to control systems, includingcomputer program (software) products, used for monitoring and/or controlof the biological processes, including monitoring and/or control of theaddition of fresh culture medium.

Particular embodiments of the invention are based on the finding thatcertain control methodologies, which may be automated, can effectivelymatch the flow rate of the CO-containing substrate to a measuredproperty of the culture medium. These methodologies, when used in aninitial operating period (e.g., a batch operation period), or when usedin general, advantageously provide a significantly improved balance interms of the reduction in acetic acid or acetate production, coupledwith the avoidance of oversupplying CO. Surprisingly, objectives of thebatch operation period or other initial operating period can be achievedmuch sooner and also much more efficiently in terms of productivities ofboth the desired end product and undesired metabolite(s), compared tothe conventional practice of establishing a CO-containing gas flow rateprofile from the outset. According to some embodiments, overall processeconomics may be greatly improved as a result of the reduced startuptime for achieving a bacteria concentration in the culture medium thatallows for transition to continuous operation. For example, the timefrom inoculation of the bioreactor until a given biomass bacteriaconcentration is achieved may be reduced by at least about 20% (e.g.,from about 20% to about 80%), typically by at least about 35% (e.g.,from about 35% to about 75%), and often by at least about 50% (e.g.,from about 50% to about 70%), compared to results achieved usingconventional practices for controlling process parameters.

According to one particular control methodology, a property of theculture medium, measured during an initial operating period (e.g., abatch operation period) or during some other operation period (e.g., acontinuous, steady-state, or normal operation period), is used as thebasis for control of the flow rate of a basic neutralizing agent (e.g.,aqueous ammonium hydroxide). Representative properties include aconcentration of an acidic metabolite (e.g., acetic acid or acetate), aproductivity of an acidic metabolite, a concentration of thecarboxydotrophic bacteria, a productivity of the carboxydotrophicbacteria, or a combination of such properties. In general, an increasein any of these properties will directionally lead to an increase in theflow rate of the basic neutralizing agent. In one specific embodiment,the basic neutralizing agent flow rate is controlled based on a targetedacidic metabolite concentration in the culture medium, which is in turndetermined from a measured concentration of the carboxydotrophicbacteria. In this manner, the control methodology accounts for theconsumption of the basic neutralizing agent, and specifically theincreased utilization of nitrogen, by the growing bacterial culture.This advantageously provides conditions during startup (e.g., a batchoperation period) that are specifically tailored to the objectives ofrapidly growing the bacterial culture with a favorable product yielddistribution.

The property of the cell culture medium may be measured continuously orintermittently, for example periodically, with the period of timebetween each successive measurement being generally from every 0.1seconds to every 120 seconds, typically from every 0.5 seconds to every60 seconds, and often from every second to every 10 seconds. Themeasured property may be obtained by on-line analysis of theconcentration, in the culture medium, of either the carboxydotrophicbacteria or the acidic metabolite. Based on successive measurements ofconcentration (e.g., in grams per liter, g/l), together with the timeinterval between successive measurements, the productivities (e.g., ingrams per liter per day, g/l·day⁻¹) of the carboxydotrophic bacteria orthe acidic metabolite can be calculated. For example if theconcentration of carboxydotrophic bacteria is determined at successiveintervals, designated Time 1 and Time 2, then the productivity of thecarboxydotrophic bacteria at Time 2 may be expressed as follows:(concentration at Time 2-concentration at Time 1)/(Time 2-Time 1).

Generally, the acidic metabolite concentration is measured in a culturemedium sample that is free or substantially free of carboxydotrophicbacteria, as a result of filtration or membrane separation. For example,a filter having a suitable pore size (e.g., in the range of 0.05 μm to 1μm) for removing the bacteria may be incorporated on a sample line of asampling system configured to withdraw cell-free culture medium from asingle reactor, or otherwise configured to withdraw such liquid frommultiple reactors (e.g., from 2 to 10 reactors, such as 4 to 6 reactors,which may operate in series or parallel, or otherwise operateindependently) at different times, in order to automatically andseparately monitor the performance of the reactors. According to otherembodiments, a cell-free sample of the culture medium may be availableas a permeate stream from a membrane separation system, in which thecell-rich retentate stream is recycled to the bioreactor. The permeate,if not used for analysis, may normally flow to a second bioreactor(e.g., operating in series). Cell-free filtrate or permeate obtainedfrom the bioreactor can provide representative samples used for theon-line measurement of the properties of end product (e.g., ethanol)concentration or acidic metabolite (e.g., acetic acid or acetate)concentration. These concentrations may be determined by knownanalytical methods, such as chromatography (e.g., high pressure liquidchromatography, or HPLC).

In the case of carboxydotrophic bacteria concentration as the measuredproperty, the culture medium may be withdrawn directly from thebioreactor, for example as a bleed stream that may normally flow to asecond bioreactor (e.g., operating in series) if not used for analysis.A sample line from a bleed stream or other stream for withdrawing cellculture medium may be fluidly connected to a suitable analytical devicefor the on-line measurement of the property of carboxydotrophic bacteriaconcentration. Representative devices include those measuring theabsorbance or transmission of electromagnetic energy through the sample(e.g., a spectrophotometer), a certain biological activity of the sample(e.g., a plate reader), or another property of the sample (e.g.,impedance/capacitance) in a disposable or reusable probe (e.g., anon-line biomass probe). The sample line from a bleed stream or otherstream may be part of a sampling system configured to withdraw culturemedium from a single reactor, or otherwise configured to withdraw suchliquid from multiple reactors (e.g., from 2 to 10 reactors, such as 4 to6 reactors, which may operate in series or parallel, or otherwiseoperate independently) at different times, in order to automatically andseparately monitor the performance of the reactors.

Sampling systems for the on-line analysis of culture media from one ormultiple bioreactors will include suitable conduits (e.g., tubing orpiping) valves, pumps, and actuators to allow the automated sampling ofa desired reactor at a desired time, and suitable devices for flushing(purging) sample lines to obtain accurate results. In the case ofanalyzing the cell-free culture medium, for example to obtain theconcentration of ethanol or acetate, filtered liquid or membranepermeate, as described above, may be fed (e.g., pumped using aperistaltic pump) at least intermittently, but preferably continuously,through a suitable sample container that is configured for on-lineanalysis. For example, inlet and outlet lines in fluid communicationwith such a sample container (e.g., a sample vial) may continuously leada filtered stream of culture medium to and from the sample container.The continuous feed of culture medium through a sample container,according to some embodiments, will involve flowing a cell-free permeateor filtrate stream, as described above, from the sample container inlet,through the sample container, and to the sample container outlet oversome period of operation of the bioreactor, for example over at leastabout 3 minutes, at least about 5 minutes, or at least about 10 minutes.According to a specific embodiment, for example, filtered, cell-freeculture medium may be fed continuously through the sample container for9 minutes, followed by a 1 minute backflush of the filter on the sampleline, in order to prevent filter plugging. Excess culture medium that isnot sampled and that flows through the sample container outlet, may bediscarded as waste.

In this manner, the liquid present in the sample container isrepresentative of the cell-containing culture medium in the bioreactor,in terms of the concentrations of the desired end product (e.g.,ethanol) and metabolite(s) (e.g., acetic acid or acetate) in thiscell-containing culture medium at the time of analysis of the cell-freeculture medium in the sample container. The lengths of the sample linesmay be minimized to minimize any offset between the actualconcentration(s) of end product and/or metabolite(s) in the bioreactorand the measured concentration(s) of the cell-free culture medium in thesample container at the time of analysis. According to some embodiments,the offset between the actual and measured concentration of the endproduct and/or a metabolite will be less than about 10%, less than about5%, or less than about 2%. A sample of the cell-free culture medium maytherefore be withdrawn from the sample container and analyzed, in orderto determine the concentration(s) of end product and metabolite(s) inthe bioreactor essentially in real time. For example, automated samplingmay involve using a sampling needle to pierce a rubber seal on the topof the sample container and withdraw a sample of cell-free culturemedium at regular intervals, with a period of time between successivemeasurements being as described above. An automated sampling apparatusmay include, for example, from 2 to 10 sample containers, such as 4 to 6sample containers, for sampling culture media from the same number ofbioreactors, which may operate in series or parallel, or otherwiseoperate independently.

More generally, automated sampling apparatuses may be configured, usingsuitable conduits (e.g., tubing or piping) valves, pumps, and actuators,for analysis of both the cell culture medium and cell-free culturemedium, as described above, of multiple reactors (e.g., from 2 to 10reactors, such as 4 to 6 reactors, which may operate in series orparallel, or otherwise operate independently) at different times, inorder to automatically and separately monitor the performance of thereactors. Properties of the culture medium, including the concentrationand productivity of metabolite(s) (e.g., acetic acid or acetate) and/orthe concentration and productivity of the carboxydotrophic bacteria, maybe determined automatically at regular intervals, with a period of timebetween successive measurements being as described above.Advantageously, the use of on-line, automated sampling and analysisallows the analytical results to be directly input to the relevantcontroller (e.g., for controlling the flow rate of the basicneutralizing agent), without human intervention. In addition, automatedsampling apparatuses as described herein allow for the monitoring ofproperties of a bioreactor culture medium, or multiple bioreactorculture media, on an essentially real-time basis, without the need foroperators to track and handle, for example by performing dilutionsand/or pipetting, multiple liquid samples from multiple bioreactors.Reliability and data reproducibility are thereby significantly improved,as well as the overall operation of the bioreactor(s).

Preferably, the control methodologies as described herein are automated,involving the use of a computer program with appropriate instructionsfor causing a processor to transmit the necessary signals to controllersfor carrying out these control methodologies. According to a particularcontrol methodology, a measured property of the culture medium is usedas the basis for controlling the flow rate of the basic neutralizingagent (e.g., a hydroxide compound, such as aqueous ammonium hydroxide orother inorganic or organic base). Such a control methodology can,compared to conventional control methodologies, advantageously reducethe time of an initial operating period (e.g., a batch operationperiod), for example prior to a period of steady-state or continuousoperation, which may be demarcated by a defined rate of withdrawal of adesired end product (e.g., ethanol) or other defined operatingparameter. Without being bound by theory, the reduction in time may beattributed at least partly to the fact that the carboxydotrophicbacteria utilize or consume the basic neutralizing agent (e.g., utilizenitrogen in the basic neutralizing agent). In general, therefore,control methodologies as described herein are particularly advantageousin bioreactor processes in which at least two feed streams (e.g., both aCO-containing substrate and a basic neutralizing agent) to the culturemedium are consumed, metabolized, or otherwise utilized by the bacteriacontained therein. In other embodiments, control methodologies describedherein may be used for both a batch operation period and a continuousoperation period, or for a continuous operation period only.

Representative properties include a measured concentration (i.e., inunits of mass/volume, such as grams/liter or grams·liter⁻¹) or ameasured productivity (i.e., in units of mass/(volume·time), such asgrams/(liter·day) or grams·liter⁻¹·day⁻¹) of the acidic metabolite(e.g., acetic acid or acetate), or of the carboxydotrophic bacteria.According to preferred embodiments, the measured property is a measuredconcentration or measured productivity of the acidic metabolite. Any ofthe above properties may be measured continuously or intermittently(e.g., periodically) during an initial operating period (e.g., a batchoperation period) or other period, with a measurement frequency, andusing sampling techniques, as described above. For example, a sample ofa permeate stream that is cell-free or at least substantially cell-free,may be analyzed for its concentration of acidic metabolite using HPLC.

Control of the flow rate of the basic neutralizing agent may, morespecifically, be based on a difference between any of the measuredproperties of the culture medium, as described above, and theircorresponding set points. For example, if an acidic metabolite measuredconcentration is the basis for control, then the basic neutralizingagent flow rate may be controlled based on the difference between theacidic metabolite measured concentration and an acidic metabolite setpoint concentration in the culture medium. Likewise, if an acidicmetabolite measured productivity, a carboxydotrophic bacteria measuredconcentration, or a carboxydotrophic bacteria measured productivity isthe basis for control, then the basic neutralizing agent flow rate maybe controlled based on the difference between (i) the acidic metabolitemeasured productivity and an acidic metabolite set point productivity,(ii) the carboxydotrophic bacteria measured concentration and acarboxydotrophic bacteria set point concentration, or (iii) thecarboxydotrophic bacteria measured productivity and a carboxydotrophicbacteria set point productivity.

In the case of an acidic metabolite set point concentration beingdetermined, for example, if the acidic metabolite measured concentrationexceeds this set point (or target) concentration, the controlmethodology may result in directionally decreasing the flow rate of thebasic neutralizing agent. This will ultimately decrease theconcentration of acidic metabolite in the culture medium, as thedecreased flow rate of basic neutralizing agent will cause the pH of theculture medium to decrease. According to preferred embodiments, the

CO-containing substrate flow rate may be controlled based on a measuredpH value (e.g., obtained using an on-line pH meter) of the culturemedium. Therefore, a decrease in the measured pH value (e.g., to below apH value set point or target, such as 4.0, 4.5, 5.0, 5.5, or 6.0) maycause an increase in the CO-containing substrate flow rate. When theculture medium becomes supplied with an increased flow of CO-containingsubstrate, acidic metabolite productivity decreases in favor of ethanolproductivity, causing the acidic metabolite concentration to decrease,e.g., directionally toward the acidic metabolite set pointconcentration, and the pH value to increase. Conversely, if the acidicmetabolite measured concentration falls below the determined set point(or target) concentration, the control methodology may result indirectionally increasing the flow rate of the basic neutralizing agent.This will ultimately increase the concentration of acidic metabolite inthe culture medium, as the increased flow rate of basic neutralizingagent will cause the pH of the culture medium to increase. TheCO-containing substrate flow rate may be controlled based on a measuredpH value (e.g., obtained using an on-line pH meter) of the culturemedium, as described above. Therefore, an increase in the measured pHvalue (e.g., to above a pH value set point or target, such as 4.2, 4.7,5.2, 5.7, or 6.2) may cause a decrease in the CO-containing substrateflow rate. When the culture medium becomes supplied with a decreasedflow of CO-containing substrate, acidic metabolite productivityincreases at the expense of ethanol productivity, causing the acidicmetabolite concentration to increase, e.g., directionally toward theacidic metabolite set point concentration, and the pH value to decrease.

Analogous control methodologies are possible, by controlling the flow ofthe basic neutralizing agent according to other measured properties ofthe culture medium, as described above. For example, (i) if the acidicmetabolite measured productivity exceeds a corresponding set point (ortarget) productivity, the control methodology may result indirectionally decreasing the flow rate of the basic neutralizing agent,(ii) if the carboxydotrophic bacteria measured concentration exceeds acorresponding set point (or target) concentration, the controlmethodology may result in directionally increasing the flow rate of thebasic neutralizing agent, or (iii) if the carboxydotrophic bacteriameasured productivity exceeds a corresponding set point (or target)concentration, the control methodology may result in directionallyincreasing the flow rate of the basic neutralizing agent. FIG. 1 depictsa representative control methodology in which the flow rate of the basicneutralizing agent, aqueous ammonium hydroxide (NH₄OH), is based on themeasured productivity of the acidic metabolite, acetic acid. The NH₄OHflow rate, in turn, affects the pH of the culture medium. If theresponse to any change in NH₄OH flow rate is maintenance of the culturemedium pH (i.e., the pH is “flat”), then the flow rate of theCO-containing substrate remains unchanged. However, if such a responseincreases the culture medium pH above its set point (i.e., pH is“high”), then the flow of the CO-containing substrate is decreased,increasing acetic acid productivity and bringing the pH back to its setpoint. If such a response decreases the culture medium pH below its setpoint (i.e., pH is “low”), then the flow of the CO-containing substrateis increased, decreasing the acetic acid productivity and bringing thepH back to its set point.

Any of the set points for properties of the culture medium (e.g., acidicmetabolite set point concentration, acidic metabolite set pointproductivity, carboxydotrophic bacteria set point concentration, orcarboxydotrophic bacteria set point productivity) may be determined, inturn, based on one or more other measured operating parameters (e.g.,measured flow rates, concentrations, and/or productivities, or pH) ofthe bioreactor process. For example, the carboxydotrophic bacteriameasured concentration or carboxydotrophic bacteria measuredproductivity may be used to determine a set point. According to aspecific embodiment, and based on certain discoveries relating to thepresent invention, the set point may be proportional to thecarboxydotrophic bacteria measured concentration or carboxydotrophicbacteria measured productivity. The acidic metabolite set pointproductivity, may, for example, be independently determined by theformulas

A ₁·BIOCONmv+B ₁ or A ₂·BIOPRODmv+B ₂

-   -   wherein A₁ and A₂ represent constants of proportionality between        the set point and the carboxydotrophic bacteria measured        concentration (BIOCONmv) or carboxydotrophic bacteria measured        productivity (BIOPRODmv), respectively, and B₁ and B₂ represent        offsets. The constants A₁ and B₁, or A₂ and B₂, may be        determined empirically from experimental data, for example prior        data obtained using the same bioreactor, or otherwise obtained        using a bioreactor containing a microbial culture for carrying        out the same conversion process (e.g., the conversion of CO to        ethanol). More specifically, these constants may be obtained by        conducting a linear regression analysis of such prior data. In        the case of determining BIOCONmv or BIOPRODmv, sampling and        analysis to determine the carboxydotrophic bacteria        concentration may be performed as described above.

In an exemplary embodiment, therefore, a carboxydotrophic bacteriameasured concentration (BIOCONmv) or carboxydotrophic bacteria measuredproductivity (BIOPRODmv) may be obtained using an on-line biomass probeor other sampling device and sample analyzer. From the value of BIOCONmvor BIOPRODmv, an acidic metabolite set point concentration (or targetconcentration) or acidic metabolite set point productivity (or targetproductivity) may be determined, for example according to the formulasgiven above.

A diluent such as fresh culture medium is generally added to thebioreactor, if not initially, then at some later point in time duringthe biological conversion process. The diluent may be first introduced,i.e., the diluent flow commenced, at the same time that one or moreother feeds to the bioreactor (e.g., the CO-containing substrate and/orthe basic neutralizing agent) are first introduced. Otherwise, thediluent may be first introduced some time after (e.g., at least about 2hours after, at least about 6 hours after, or at least about 12 hoursafter) one or more other feeds to the bioreactor (e.g., theCO-containing substrate and/or the basic neutralizing agent) are firstintroduced. The fresh culture medium flow may be commenced afterattaining a suitable culture medium commencement target, which may bethe same as any of the process initiation targets as described above.Such a target may include, for example, a predetermined concentration orproductivity of either the carboxydotrophic bacteria or the acidicmetabolite. In general, the addition of a diluent, such as fresh culturemedium, at a given mass flow rate or volumetric flow rate is accompanied(e.g., simultaneously) by the withdrawal of culture medium, includingthe desired end product and any metabolites, at a comparable mass flowrate or volumetric flow rate. The withdrawn culture medium may (i) befree or substantially free of carboxydotrophic bacteria (e.g., in thecase of being separated by filtration or membrane separation), or (ii)contain carboxydotrophic bacteria in the same or substantially the sameconcentration as in the culture medium contained in the bioreactor(e.g., in the case of being withdrawn without separation). In somecases, the withdrawn culture medium may include portions (e.g., separatestreams) of both (i) and (ii). In any event, either or both of (i) and(ii) may be fed to a second bioreactor for carrying out the samebiological CO to ethanol conversion process (e.g., by operating inseries with the first bioreactor).

Preferably, the flow rate of the diluent is increased gradually duringall or part of a batch operation period as defined herein. However, itis not required that any diluent flow be added during this period, suchthat diluent flow is added only during a later (e.g., continuous)operation period, or such that the introduction of diluent to thebioreactor is used to demarcate the transition from a batch operatingperiod to a continuous operating period.

As with the basic neutralizing agent flow rate, the diluent flow ratemay be controlled based on any of the measured properties of the culturemedium, and using any of the control methodologies, as described above.According to particular embodiments, the diluent flow rate to thebioreactor is controlled based on the carboxydotrophic bacteria measuredconcentration or carboxydotrophic bacteria measured productivity in theculture medium. Based on certain discoveries relating to the presentinvention, a diluent flow rate set point may be determined according toan exponential function, with the measured concentration or measuredproductivity being the exponent. For example, the diluent flow rate setpoint may be determined according to one of the formulas

C ₁ ^((BIOCONmv)) or C ₂ ^((BIOPRODmv))

-   -   wherein BIOCONmv and BIOPRODmv represent, respectively, the        carboxydotrophic bacteria measured concentration and the        carboxydotrophic bacteria measured productivity, respectively,        and C₁ and C₂ are constants. The constants C₁ and C₂ may be        determined empirically from experimental data, for example from        prior data obtained using the same bioreactor, or otherwise        obtained using a bioreactor containing a microbial culture for        carrying out the same conversion process (e.g., the conversion        of CO to ethanol). In the case of determining BIOCONmv or        BIOPRODmv, sampling and analysis to determine the        carboxydotrophic bacteria concentration may be performed as        described above.

According to a second particular control methodology, measuring aproperty of the culture medium is not required. Rather, prior data maybe used to establish a relationships among the variables ofcarboxydotrophic bacteria concentration and productivity, and thecorresponding flow of CO-containing gas (or substrate) of a givencomposition that will provide a targeted productivity of the acidicmetabolite, as well as the flow of basic neutralizing agent that willmaintain the pH of the culture medium. The prior data may be obtained,for example, using the same bioreactor, or otherwise using a bioreactorcontaining a microbial culture for carrying out the same conversionprocess (e.g., the conversion of CO to ethanol). By using informationfrom other biological CO-to-ethanol conversion processes, includingcarboxydotrophic bacteria concentration and productivity, in addition tothe corresponding flow rate of CO-containing substrate, the flow rate ofthe basic neutralizing agent may be estimated for a desired acidicmetabolite productivity. Furthermore, using such information, the flowrate of CO-containing substrate can be estimated to supply a givencarboxydotrophic bacteria concentration and achieve the desired acidicmetabolite productivity.

Specific relationships among the process variables may be based, forexample, on the equations below:

W·BIOPROD+X·METPROD=NEUTFLO=Y·COFLO+Z

-   -   wherein BIOPROD, METPROD, NEUTFLO, and COFLO represent,        respectively, the carboxydotrophic bacteria productivity, the        acidic metabolite productivity, the basic neutralizing agent        flow rate to the bioreactor, and the CO-containing substrate        flow rate to the bioreactor, and W, X, Y, and Z are constants        that are determined empirically based on prior data, as        described above. More specifically, these constants may be        obtained by conducting a linear regression analysis of such        prior data. The productivities can be measured as described        above (e.g., using a spectrophotometer, plate reader, or biomass        probe in the case of carboxydotrophic bacteria concentration or        productivity, and/or using HPLC in the case of acidic metabolite        concentration or productivity).

According to particular embodiments, therefore, during a batch operatingperiod, or other operating period, of feeding both the CO-containingsubstrate and basic neutralizing agent to the bioreactor, the basicneutralizing agent flow rate is controlled based on the flow rate of theCO-containing substrate. For example, the basic neutralizing agent flowrate may be controlled based on either a measured value (i.e., aCO-containing substrate measured flow rate) or otherwise a set pointvalue (i.e., a CO-containing substrate flow rate set point). That is, aset point for the basic neutralizing agent flow rate may be determinedaccording to such a measured value or set point value. According tocertain embodiments, as is apparent from the process variablerelationships set forth above, the basic neutralizing agent flow rateset point may vary linearly with either the CO-containing substratemeasured flow rate or CO-containing substrate flow rate set point. Stillmore specifically, the basic neutralizing agent flow rate set point maybe determined according to the formulas:

Y·COFLOmv+Z or Y·COFLOsp+Z

-   -   wherein COFLOmv and COFLOsp represent, respectively, the        CO-containing substrate measured flow rate and the CO-containing        substrate flow rate set point. Y and Z represent constants,        namely a constant of proportionality between COFLOmv or COFLOsp        and the basic neutralizing agent flow rate set point, in the        case of Y, and an offset, in the case of Z.

In particular types of these control methodologies, the flow rate of theCO-containing substrate may be, in turn, controlled based on the pHvalue of the culture medium. For example, if the pH measured value ofthe culture medium falls below a pH set point (e.g., one of the specificpH values indicated above), the culture medium has become too acidic,and, in response, the CO-containing substrate flow rate is increased(e.g., by automatically increasing a percentage opening of a controlvalve on a CO-containing substrate inlet line) to supply more CO to thebacteria culture and reduce the productivity of acid metabolite.Conversely, if the pH measured value of the culture medium rises abovethis pH set point, the culture medium has become too basic, and, inresponse, the CO-containing substrate flow rate is decreased (e.g., byautomatically decreasing a percentage opening of a control valve on aCO-containing substrate inlet line) to supply less CO to the bacteriaculture and increase the productivity of acid metabolite.

Alternatively, a CO-containing flow rate set point may be determinedfrom a measured pH value of the culture medium, with this set pointrepresenting a deviation from the CO-containing flow rate measuredvalue. In view of these considerations, it may be possible for themeasured pH value of the culture medium to generate the set points forboth the flow rate of the CO-containing substrate in addition to theflow rate of the basic neutralizing agent. However, it is generallypreferred that the CO-containing substrate measured flow rate, thismeasured flow rate (as opposed to the flow rate set point) is used todetermine the set point for the basic neutralizing agent flow rate. Theculture medium pH value may be measured either continuously orintermittently (e.g., periodically at regular intervals) using, forexample, an on-line pH analyzer. Otherwise, this pH value may bemeasured manually.

EXAMPLES

The following examples are set forth as representative of the presentinvention. These examples are not to be construed as limiting the scopeof the invention as these and other equivalent embodiments will beapparent in view of the present disclosure and appended claims.

Example 1 Comparison of Conventional “Time-Based” Startup and Inventive“Automated” Startup

A biological process for the conversion CO to ethanol was started byinoculating a bioreactor with culture medium containing C. ljundahlii.The pH of culture medium began to drop as acetic acid was produced.CO-containing substrate and ammonium hydroxide feeds to the bioreactorwere started when the pH of the culture medium reached 5.0. The flowrate of the CO-containing substrate over the startup was governed by aconventional, predetermined time-based profile, in which the avoidanceof CO oversupply was the main objective. For comparative purposes, thesame process was started using a control methodology as describedherein, in which the flow rate of the ammonium hydroxide was controlledbased on the concentration of acetate (in the form of acetic acid) inthe culture medium, measured automatically and periodically by HPLC. Theprogress of these comparative startups is shown in FIGS. 2 and 3, whichprovide the concentrations of ethanol, bacteria, and acetic acid in theculture medium over a period of two days. This information is providedin the case of the conventional, time-based startup (FIG. 2—“Time-BasedControl”) and in the case of the automated startup (FIG. 3—“AutomatedControl”), according to a representative embodiment of the invention.

As is apparent from a comparison of FIGS. 2 and 3, the concentration ofthe desired product, ethanol, is less than 2 grams/liter (g/l) at Day 1of the time-based startup, whereas this concentration is already nearly8 g/l at this point in the automated startup. In addition, asillustrated in FIG. 4, it is apparent that the automated startup leadsto an increase in the CO-containing substrate flow rate that is muchfaster, compared to the time-based start-up. This is due to thecontinual supply of the needed amount of CO to the bacterial culture forethanol production, without oversupply that is detrimental to bacterialgrowth. In the case of the time-based profile, the flow rate of theCO-containing substrate was characteristically conservative, in order toensure CO oversupply is avoided. As a result, however, CO undersupply isinevitable, and acetic acid is main product, rather than ethanol. FIG. 5compares the concentrations of bacteria over time, for these start-upprocesses, using these two control methodologies. As is apparent, evenwith the higher CO flow rate in the case of the automated start-up,microbial growth is not inhibited, and in fact it is enhanced.

Based on these results, control methodologies as described herein canprovide significant process benefits, particularly in terms of reducingthe time needed to achieve a given process objective, such as a desiredacetic acid concentration or bacteria concentration. The objective maybe associated with the completion of an initial startup period, such asa batch operation period, in which case the transition to continuousoperation may be attained more quickly and efficiently. This leads toimportant commercial benefits, including a reduced consumption ofmaterials and reduced overall operating costs. In the case of a processoperating with two reactors equipped with a cell recycle system, it maybe possible to directly sample cell-free permeate from the reactors andfeed these samples to an automated HPLC without any further treatment,i.e., without sample filtration or centrifugation. In contrast,conventional sample preparation methods, prior to injection to an HPLC,require the addition of specific acids or bases, followed bycentrifugation or filtration. This involves manual pipetting, which addscomplexity and results in greater error in the results.

Example 2 Automated Startup Control of NH₄OH Flow Based on MeasuredConcentrations

A biological process for the conversion CO to ethanol was started byinoculating a bioreactor with culture medium containing C. ljundahlii.The pH of culture medium began to drop as acetic acid was produced.CO-containing substrate and ammonium hydroxide feeds to the bioreactorwere started when the pH of the culture medium reached 5.0. Based on themeasured bacteria concentration in the bioreactor, an acetate (aceticacid) target concentration and a diluent flow rate were determinedaccording to the following equations:

Acetate target concentration=A ₁·BIOCONmv+B ₁ Diluent flow rate=C ₁^((BIOCONmv))

Wherein A₁, B₁, and C₁ were determined empirically from informationobtained in prior processes. Based on the acetic acid concentrationmeasured using the on-line HPLC, the flow rate of ammonium hydroxide wasadjusted automatically, i.e., increased in order to increase the aceticacid production by the bacteria or decreased in order to decrease theacetic acid production. The flow of the gaseous CO-containing substratewas increased or decreased automatically in order to maintain the pH ofthe culture medium at target pH=5.0. The concentrations of ethanol,bacteria, and acetic acid over time, in addition to the flow rate of thediluent, are shown in FIG. 6.

Example 3 Automated Startup Based on Measured pH and CO-ContainingSubstrate Flow Only

Based on previous start-up data for biological processes, as describedin Example 1, in which CO was converted to ethanol by feeding it to aculture medium containing C. ljundahlii, relationships were establishedbetween a given bacterial concentration in the reactor, a correspondingflow rate of the CO-containing substrate of a given composition requiredthat to yield a target acetic acid productivity, and a required ammoniumhydroxide flow rate needed to maintain the culture medium pH at a giventarget. These relationships were as follows:

W·BIOPROD+X·METPROD=NEUTFLO=Y·COFLO+Z

-   -   wherein BIOPROD, METPROD, NEUTFLO, and COFLO represented,        respectively, the bacteria (biomass) productivity, the acetic        acid (acetate) productivity, the NH₄OH flow rate to the        bioreactor, and the CO-containing substrate flow rate to the        bioreactor. The factors W, X, Y and Z were determined        empirically (using linear regression) from information obtained        in prior processes, in which bacteria productivity measurements        were based on concentrations measured at successive time        intervals. That is, the measured bacterial productivity was        calculated as bacteria concentration at Time 2-bacteria        concentration at Time 1)/(Time 2-Time 1). In these prior        processes, the bacteria concentration was measured using a        spectrophotometer or plate reader or biomass probe, and the        measured acetic acid productivity was calculated as acetic acid        concentration at Time 2-acetic acid concentration at Time        1)/(Time 2-Time 1). Acetic acid and ethanol concentrations were        measured by HPLC. According to the data generated from these        prior processes, the following factors were determined: W=1.2,        X=1.5, Y=1.46, and Z=3.21.

Thus, the relationship used for the automated startup wasNEUTFLO=1.46·COFLO+3.21. The pH of the culture medium was maintained at5.0 by adjusting the flow of the CO-containing substrate automaticallyusing a PID controller. The relationships above were used to set theammonium hydroxide flow rate, based on the measured flow rate of theCO-containing substrate.

The concentrations of ethanol, bacteria, and acetic acid over time, inaddition to the flow rates of the ammonium hydroxide and CO-containingsubstrate, are shown in FIG. 7. Advantageously, the bacterial growthover the first day was high, at about 2.9 grams/(liter·day), and aceticacid productivity was low, at about 2.8 grams/(liter·day). Ethanolproductivity and concentration were maximized. These observations wereconsistent with a successful startup of the biological CO conversionprocess, which is critical prior to establishing a continuous process.Importantly, the measured concentrations of bacteria and acetic acid inthe bioreactor were not used directly in this control methodology.Rather, these concentrations were monitored, only to the extent toconfirm progress of the operation, but without feedback into theautomation.

Overall, aspects of the invention are directed to control methodologiesfor biological fermentation processes in which a CO-containing substrateis used to produce higher value products such as ethanol. The controlmethodologies may advantageously shorten the initiation or startup ofthese processes, such that continuous production is attained (e.g., uponreaching a given process initiation target) in a shorter time periodafter inoculation of the bioreactor, compared to time periods requiredusing conventional control methodologies (e.g., a time-based profile forthe flow of CO-containing substrate). These control methodologies mayalternatively, or in addition, improve the productivities of the desiredend product and/or improve the growth rate of the bacteria, during theinitiation or startup. Those having skill in the art, with the knowledgegained from the present disclosure, will recognize that various changescan be made in control methodologies, systems, and computer programproducts, without departing from the scope of the present invention.

1. A computer program product, comprising a non-transitory computerreadable medium having a computer program embodied thereon, the computerprogram including instructions for causing a processor to perform thesteps of: receiving, from an analyzer configured to analyze a sample ofa culture medium from a bioreactor, a measured property of the culturemedium, and inputting, to a controller configured to control a basicneutralizing agent flow rate to the bioreactor, the measured property ofthe culture medium as a basis for control, wherein the measured propertyis selected from the group consisting of an acidic metabolite measuredconcentration, an acidic metabolite measured productivity, acarboxydotrophic bacteria measured concentration, a carboxydotrophicbacteria measured productivity, or a combination thereof.
 2. A computerprogram product, comprising a non-transitory computer readable mediumhaving a computer program embodied thereon, the computer programincluding instructions for causing a processor to perform the steps of:receiving a CO-containing substrate measured flow rate to a bioreactoror a CO-containing substrate flow rate set point, inputting, to acontroller configured to control a basic neutralizing agent flow rate tothe bioreactor, the CO-containing substrate measured flow rate or theCO-containing substrate flow rate set point as a basis for control. 3.The computer program product of claim 2, wherein the computer programincludes instructions for causing the processor to perform the furthersteps of: receiving a measured pH value, and inputting, to a secondcontroller configured to control a CO-containing substrate flow rate,the measured pH value as a basis for control.
 4. The computer programproduct of claim 2, wherein the measured pH value is received from ananalyzer configured to analyze a sample of a culture medium from thebioreactor.
 5. A system comprising: a bioreactor, a controllerconfigured to control a basic neutralizing agent flow rate to thebioreactor, based on a CO-containing substrate measured flow rate to thebioreactor or a CO-containing substrate flow rate set point.
 6. Thesystem of claim 5, further comprising a second controller configured tocontrol a CO-containing substrate flow rate based on a measured pHvalue.
 7. The system of claim 6, further comprising: a samplingapparatus configured to isolate, from the bioreactor, a sample of aculture medium, and an analyzer configured to analyze the sample andinput, to the controller, the measured pH value.